Project Summary Addressing the objective of PAR-16-355 to ?expand approaches for understanding epigenetic mechanisms by which social factors lead to biological changes that affect health disparities,? our study will conduct novel population-based discovery and replication analyses to investigate how DNA methylation (DNAm) varies with exposure to racial discrimination, economic hardship, and air pollution, and implications for disparities in cardiometabolic disease risk and accelerated aging (defined as epigenetic age > chronological age). For the discovery phase, we will newly obtain DNAm from the stored blood spots of our My Body, My Story (MBMS) study (R01 AG027122), whose participants comprise a random sample of 1005 US-born non- Hispanic black (n = 504) and non-Hispanic white (n = 501) adults, aged 35 to 64 years, recruited from four community health centers in Boston, MA (2008-2010). This study has rich data on: (a) racial discrimination and socioeconomic position, each measured at multiple levels and across the life-course, plus residential air pollution in the past year; (b) cardiometabolic outcomes; and (c) relevant covariates. For replication, we will use the newly available analogous DNAm, exposure, and health data from a subset of Wave 5 (2010-2012) participants in the Multi-Ethnic Study of Atherosclerosis (MESA) (N = 1264, age 55-94; 582 non-Hispanic white, 270 non-Hispanic black; 404 Hispanic, from Baltimore, MD, Forsyth County, NC, NYC, NY, and St. Paul, MN). To strengthen causal inference based on the cross-sectional data, we will employ MR-Base, a recently constructed highly powerful application that harnesses publicly-available genome-wide association study (GWAS) summary data and Mendelian Randomization (MR) to assess causal directions of associa- tions between exposures, DNAm, and health outcomes. Our Specific Aims thus are: Aim 1: Conduct novel analyses to identify variation in DNAm associated with: (1) Aim 1.1: exposure to racial discrimination, economic hardship, and air pollution; and (2) Aim 1.2: measured cardiometabolic outcomes (blood pressure, fasting insulin, Type 2 diabetes, Framingham Cardiovascular Disease 10-year risk score, metabolic syndrome); plus (3) Aim 1.3: For DNAm-health outcome associations observed in Aim 1.2, use MR-Base to strengthen causal inference about the direction of the associations (e.g., methylation causes vs. is due to disease); discovery analyses: MBMS; replication analyses: MESA. Aim 2: Assess the relationships between both the Aim 1 study exposures and health outcomes with accelerated aging (epigenetic age > chronological age), as identified by three newly identified DNAm ?clocks? (Horvath, Hannum, and DNAm PhenoAge); discovery: MBMS; replication: MESA. Aim 3: Analyze if the methylation sites and ?clocks? associated with both the study exposures and health outcomes mediate these exposure-outcomes associations, thereby contributing to health inequities. Impact: Results will advance knowledge about DNAm mechanisms contributing to health disparities.